文冠果性别分化的内源激素与microRNA调控机制

Few bisexual flowers and many male flowers is the main factor that leads to low seed yield in Xanthoceras sorbifolia. Sex organ abortion leads to sex differentiation in the late development stage. Endogenous hormone and microRNA play important regulatory role in sex differentiation, and constitute interaction network to control sexual organ morphogenesis. Previously, we have observed flower bud differentiation, identified flower bud microRNA of different flower types, observed endogenous hormone of bisexual and male flower in florescence. In this study, we will focus on pistil and stamen development of bisexual and male flowers, establish association between environmental condition, flower bud external morphology and internal structure development. To identify the key endogenous hormone and hormone balance that affect sex differentiation, hormone location and quantitative analysis will be used to analyze the endogenous hormone distribution characters and variation on the organ, tissue, cellular level. MicroRNA expression differences and target genes functions will be analyzed at critical time during pistil and stamen development. For the microRNA and target genes related with hormone synthesis, signal transduction, pistil and stamen development, their spatio-temporal expression patterns and the relationship with each hormone will be identified. MicroRNA, hormone and their interactions on sex differentiation will be illuminated. The results of this study will provide important theory basis for enhance bisexual flower proportion and seed yield using molecule and physiology techniques.

两性花少、雄花多是导致文冠果（Xanthoceras sorbifolia）种子低产的主要因素。两性花和雄花是分化后期性器官败育导致性别分化。内源激素和microRNA对性别分化具有重要调控作用，二者构成互作网络影响性器官形态建成。本项目在已开展花芽分化观测、不同类型花芽microRNA研究的基础上，关注两性花和雄花雌雄蕊发育过程，建立环境条件、花芽外部形态与内部组织结构的关联；通过激素定位与定量分析，研究花芽分化期各内源激素在细胞、组织、器官水平的分布和变化规律，确定影响性别分化的关键激素及激素平衡关系；明确雌雄蕊发育关键时间点microRNA表达差异和靶基因功能，分析与激素合成和信号转导、雌雄蕊发育有关的microRNA及靶基因的时空表达模式、与内源激素的关系，阐明microRNA与激素对性别分化的影响。研究结果将为利用生理、分子等技术提高两性花比例，进而提高产量提供理论依据。

文冠果为雌雄同株异花树种，雄能花数量远多于雌能花导致其种实产量低，通过科学的方法调控其雌雄比例成为了解决这一问题的有效手段。本项目确立了文冠果花芽分化过程中花芽内部结构与外部形态之间稳定的对应关系，明确了雌、雄蕊败育的发生过程。雌能花中的雌蕊发育正常，雄能花中的雌蕊停止生长并有萎缩趋势，柱头上没有形成乳突状细胞。雄能花中的雄蕊正常，雌能花中的花粉开始呈现液泡化衰败；通过监测气象数据，建立了气象因子与文冠果花芽分化之间的关系。当≥K（生物学零度）有效积温达到1402.4℃时，花原基出现，花芽开始分化。当≥K有效积温达到2808.4℃时，雄蕊原基出现。次年3月初，≥K有效积温达到20.2℃时，雌蕊原基开始分化。当≥K有效积温达到230.6℃时，雄能花的雌蕊开始发生败育。当≥K有效积温达到264℃时，雌能花的花粉出现液泡化衰败。当≥K有效积温达到311.8℃时，雄能花的雌蕊彻底败育。当≥K有效积温达到353.1℃时，雌能花的雄蕊败育完全。利用高效液相色谱法（HPLC）明确了内源激素对文冠果花芽分化的影响。高水平的ZT、IAA和ABA利于雌能花的发育。高水平的GA具有促雄效应。高水平的ABA/IAA、ABA/GA、ZT/GA有利于花芽分化。高水平的ABA/IAA、ZT/IAA对雄蕊发育有利。通过喷施植物生长调节剂，探究了6-BA和GA3对文冠果开花结实的作用。喷施160mg/L 6-BA可增加顶花序雌能花的比例，提高果实和种子品质。喷施150mg/L GA3可提高坐果率。喷施6-BA和GA3均可提高顶花序中蛋白质含量。根据文冠果性别分化的关键时期，利用第二代高通量测序技术构建了2个转录组及4个microRNA文库，挖掘出6个MADS-box相关基因及直接影响文冠果的雌雄蕊发育、激素合成和信号转导的28个miRNA；明确了适用于文冠果性别分化的最优内参基因组合（UBQ和eIF-4α）。本项目阐述了文冠果花芽分化过程中内源激素及miRNA对其性别分化的影响及相互联系，为后续探究关键基因对文冠果雌蕊发育的作用机制奠定了基础。